RESUMO
Matrix metalloproteinases (MMPs) play important roles in wound healing, but attribution of their functions in repair of wounds has been challenging. Commonly used tools such as MMP-knockout mice and zymography often confound analysis, which is complicated further as these enzymes exist in three distinct forms with only one being catalytically competent. With the use of topical exogenously administered recombinant MMP-8 and MMP-13 to diabetic and nondiabetic mouse wounds, we show that these proteinases facilitate wound repair by upregulating IL-6 and increasing neutrophil trafficking with an early onset of inflammation. Furthermore, by spatiotemporal control in the use of a selective MMP-2 inhibitor, along with immunoprecipitation and Western blotting, we provide definitive demonstration that MMP-2 does not affect wound healing, contrary to reports. MMP-2 is found in wounds complexed with TIMPs, which is catalytically incompetent.
RESUMO
Active vaccination examining a single hapten engendered with a series of peptidic linkers has resulted in the production of antimethamphetamine antibodies. Given the limited chemical complexity of methamphetamine, the structure of the linker species embedded within the hapten could have a substantial effect on the ultimate efficacy of the resulting vaccines. Herein, we investigate linker effects by generating a series of methamphetamine haptens that harbor a linker with varying amino acid identity, peptide length, and associated carrier protein. Independent changes in each of these parameters were found to result in alterations in both the quantity and quality of the antibodies induced by vaccination. Although it was found that the consequence of the linker design was also dependent on the identity of the carrier protein, we demonstrate overall that the inclusion of a short, structurally simple, amino acid linker benefits the efficacy of a methamphetamine vaccine in limiting brain penetration of the free drug.
Assuntos
Estimulantes do Sistema Nervoso Central , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Haptenos/imunologia , Metanfetamina , Adjuvantes Imunológicos/química , Animais , Anticorpos , Afinidade de Anticorpos , Especificidade de Anticorpos , Sistema Nervoso Central/imunologia , Estimulantes do Sistema Nervoso Central/química , Estimulantes do Sistema Nervoso Central/imunologia , Estimulantes do Sistema Nervoso Central/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Toxoide Diftérico/química , Toxoide Diftérico/imunologia , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Haptenos/química , Metanfetamina/química , Metanfetamina/imunologia , Metanfetamina/metabolismo , Metanfetamina/farmacologia , Camundongos , RadioimunoensaioRESUMO
MT1-MMP and MMP2 have been implicated as pro-tumorigenic and pro-metastatic factors in a wide variety of cancers including melanoma. We have previously demonstrated that MT1-MMP is highly expressed in melanoma where it promotes melanoma cell invasion and metastasis in part through the activation of its target MMP2. Given the accessibility of MMPs, as they are either secreted (e.g. MMP2) or membrane-tethered (e.g. MT1-MMP), they represent ideal targets for specific inhibition via small molecules. Here we show that the novel small-molecule inhibitor ND-322 with high selectivity for MT1-MMP and MMP2, effectively inhibits MT1-MMP and MMP2 activity resulting in reduced in vitro melanoma cell growth, migration and invasion. Importantly, these inhibitory effects lead to significant reduction of melanoma tumor growth and metastasis. We further show that while cell migration and invasion could be similarly hampered by specific inhibition of either MT1-MMP or MMP2 via shRNAs, the growth inhibitory activity of ND-322 could only be mirrored by specific inhibition of MT1-MMP. These data support ND-322 as a novel effective inhibitor capable of counteracting both MT1-MMP and MMP2, two key proteases involved in melanoma growth and metastasis. ND-322 may therefore represent a new inhibitor in the repertoire of treatments against melanoma.
Assuntos
Arginina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Melanoma/tratamento farmacológico , Metástase Neoplásica/tratamento farmacológico , Sulfetos/farmacologia , Sulfonas/farmacologia , Animais , Arginina/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Masculino , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Cysteine protease Cwp84 is responsible for surface-layer processing in Clostridium difficile and was also shown to cleave several human extracellular matrix components in vitro. To enable the facile identification and characterization of Cwp84 inhibitors, we developed a fluorogenic 10-mer peptide based on the enzyme's natural substrate SlpA that is amenable for use in FRET-based high-throughput screening. The design of substrate-mimetic inhibitors led to epoxysuccinate 8c, which displayed an inactivation efficiency (kinact/KI) of (4.7 ± 0.3) × 10(4) M(-1) min(-1). Further evaluation of 8c demonstrated its ability to inhibit fibronectin cleavage and, more importantly, subvert surface-layer biogenesis in C. difficile.
Assuntos
Clostridioides difficile/enzimologia , Cisteína Endopeptidases/metabolismo , Enterocolite Pseudomembranosa/microbiologia , Inibidores Enzimáticos/química , Glicoproteínas de Membrana/metabolismo , Peptídeos/química , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Cisteína Endopeptidases/genética , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Glicoproteínas de Membrana/genética , Peptídeos/farmacologiaRESUMO
Prolonged use of broad-spectrum antibiotics disrupts the indigenous gut microbiota, which consequently enables toxigenic Clostridium difficile species to proliferate and cause infection. The burden of C. difficile infections was exacerbated with the outbreak of hypervirulent strains that produce copious amounts of enterotoxins and spores. In recent past, membrane-active agents have generated a surge of interest due to their bactericidal property with a low propensity for resistance. In this study, we capitalized on the antimicrobial property and low oral bioavailability of salicylanilide anthelmintics (closantel, rafoxanide, niclosamide, oxyclozanide) to target the gut pathogen. By broth microdilution techniques, we determined the MIC values of the anthelmintics against 16 C. difficile isolates of defined PCR-ribotype. The anthelmintics broadly inhibited C. difficile growth in vitro via a membrane depolarization mechanism. Interestingly, the salicylanilides were bactericidal against logarithmic- and stationary-phase cultures of the BI/NAP1/027 strain 4118. The salicylanilides were poorly active against select gut commensals (Bacteroides, Bifidobacterium and Lactobacillus species), and were non-hemolytic and non-toxic to mammalian cell lines HepG2 and HEK 293T/17 within the range of their in vitro MICs and MBCs. The salicylanilide anthelmintics exhibit desirable properties for repositioning as anti-C. difficile agents.
Assuntos
Anti-Helmínticos/farmacologia , Clostridioides difficile/efeitos dos fármacos , Animais , Anti-Helmínticos/química , Anti-Infecciosos/farmacologia , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/isolamento & purificação , Células HEK293 , Hemólise/efeitos dos fármacos , Células Hep G2 , Humanos , Cinética , Potenciais da Membrana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Salicilanilidas/química , Salicilanilidas/farmacologia , Ovinos , Fatores de Tempo , Virulência/efeitos dos fármacosRESUMO
Reported herein is that (4S)-4,5-dihydroxy-2,3-pentanedione (DPD) can undergo a previously undocumented non-enzymatic glycation reaction. Incubation of DPD with viral DNA or the antibiotic gramicidinâ S resulted in significant biochemical alterations. A protein-labeling method was consequently developed that facilitated the identification of unrecognized glycation targets of DPD in a prokaryotic system. These results open new avenues toward tracking and understanding the fate and function of the elusive quorum-sensing signaling molecule.
Assuntos
Glucose/química , Percepção de Quorum , Transdução de Sinais , DNA/química , Pentanos/químicaRESUMO
The gelatinases, matrix metalloproteinases (MMP)-2 and MMP-9, are thought to be key mediators of secondary damage in adult animal models of brain injury. Moreover, an acute increase in these proteases in plasma and brain extracellular fluid of adult patients with moderate-to-severe traumatic brain injuries (TBIs) is associated with poorer clinical outcomes and mortality. Nonetheless, their involvement after TBI in the pediatric brain remains understudied. Using a murine model of TBI at postnatal day 21 (p21), approximating a toddler-aged child, we saw upregulation of active and pro-MMP-9 and MMP-2 by gelatin zymography at 48 h post-injury. We therefore investigated the role of gelatinases on long-term structural and behavioral outcomes after injury after acute inhibition with a selective gelatinase inhibitor, p-OH SB-3CT. After systemic administration, p-OH SB-3CT crossed the blood-brain barrier at therapeutically-relevant concentrations. TBI at p21 induced hyperactivity, deficits in spatial learning and memory, and reduced sociability when mice were assessed at adulthood, alongside pronounced tissue loss in key neuroanatomical regions. Acute and short-term post-injury treatment with p-OH SB-3CT did not ameliorate these long-term behavioral, cognitive, or neuropathological deficits as compared to vehicle-treated controls, suggesting that these deficits were independent of MMP-9 and MMP-2 upregulation. These findings emphasize the vulnerability of the immature brain to the consequences of traumatic injuries. However, early upregulation of gelatinases do not appear to be key determinants of long-term recovery after an early-life injury.
Assuntos
Barreira Hematoencefálica/metabolismo , Lesões Encefálicas/tratamento farmacológico , Compostos Heterocíclicos com 1 Anel/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/metabolismo , Sulfonas/metabolismo , Animais , Transporte Biológico , Lesões Encefálicas/diagnóstico , Lesões Encefálicas/enzimologia , Lesões Encefálicas/genética , Pré-Escolar , Modelos Animais de Doenças , Expressão Gênica , Compostos Heterocíclicos com 1 Anel/farmacologia , Humanos , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Inibidores de Metaloproteinases de Matriz/farmacologia , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Prognóstico , Recuperação de Função Fisiológica/fisiologia , Isolamento Social , Memória Espacial , Sulfonas/farmacologia , Índices de Gravidade do Trauma , Falha de TratamentoRESUMO
Nonhealing chronic wounds are major complications of diabetes resulting in >70,000 annual lower-limb amputations in the United States alone. The reasons the diabetic wound is recalcitrant to healing are not fully understood, and there are limited therapeutic agents that could accelerate or facilitate its repair. We previously identified two active forms of matrix metalloproteinases (MMPs), MMP-8 and MMP-9, in the wounds of db/db mice. We argued that the former might play a role in the body's response to wound healing and that the latter is the pathological consequence of the disease with detrimental effects. Here we demonstrate that the use of compound ND-336, a novel highly selective inhibitor of gelatinases (MMP-2 and MMP-9) and MMP-14, accelerates diabetic wound healing by lowering inflammation and by enhancing angiogenesis and re-epithelialization of the wound, thereby reversing the pathological condition. The detrimental role of MMP-9 in the pathology of diabetic wounds was confirmed further by the study of diabetic MMP-9-knockout mice, which exhibited wounds more prone to healing. Furthermore, topical administration of active recombinant MMP-8 also accelerated diabetic wound healing as a consequence of complete re-epithelialization, diminished inflammation, and enhanced angiogenesis. The combined topical application of ND-336 (a small molecule) and the active recombinant MMP-8 (an enzyme) enhanced healing even more, in a strategy that holds considerable promise in healing of diabetic wounds.
Assuntos
Complicações do Diabetes , Inibidores de Proteases/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Metaloproteinase 8 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Ferimentos e Lesões/enzimologiaRESUMO
SB-3CT is a potent and selective inhibitor of matrix metalloproteinase (MMP)-2 and -9, which has shown efficacy in an animal model of severe traumatic brain injury (TBI). However, SB-3CT is poorly water-soluble and is metabolized primarily to p-hydroxy SB-3CT (2), a more potent inhibitor than SB-3CT. We synthesized the O-phosphate prodrug (3) of compound 2 to enhance its water solubility by more than 2000-fold. The prodrug 3 was a poor MMP inhibitor, but readily hydrolyzed to the active 2 in human blood. Pharmacokinetics and brain distribution studies in mice showed that 2 crossed the blood-brain barrier (BBB) and achieved therapeutic concentrations in the brain. The prodrug 3/compound 2 was evaluated in a mouse model of severe TBI and found to significantly decrease the brain lesion volume and improve neurological outcomes. MMP-9 inhibition by a water-soluble thiirane inhibitor is a promising therapy for treatment of TBI.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/patologia , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Inibidores de Metaloproteinases de Matriz/uso terapêutico , Sulfonas/uso terapêutico , Animais , Área Sob a Curva , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Lesões Encefálicas/fisiopatologia , Linhagem Celular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Compostos Heterocíclicos com 1 Anel/farmacologia , Concentração Inibidora 50 , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Exame Neurológico , Desempenho Psicomotor/efeitos dos fármacos , Solubilidade , Sulfonas/farmacologia , Água/metabolismoRESUMO
The emergence of antibiotic resistance places a sense of urgency on the development of alternative antibacterial strategies, of which targeting virulence factors has been regarded as a "second generation" antibiotic approach. In the case of Pseudomonas aeruginosa infections, a proteolytic virulence factor, LasB, is one such target. Unfortunately, we and others have not been successful in translating in vitro potency of LasB inhibitors to in vivo efficacy in an animal model. To overcome this obstacle, we now integrate in silico and in vitro identification of the mercaptoacetamide motif as an effective class of LasB inhibitors with full in vivo characterization of mercaptoacetamide prodrugs using Caenorhabditis elegans. We show that one of our mercaptoacetamide prodrugs has a good selectivity profile and high in vivo efficacy, and confirm that LasB is a promising target for the treatment of bacterial infections. In addition, our work highlights that the C. elegans infection model is a user-friendly and cost-effective translational tool for the development of anti-virulence compounds.
Assuntos
Proteínas de Bactérias/metabolismo , Caenorhabditis elegans/microbiologia , Metaloendopeptidases/metabolismo , Pseudomonas aeruginosa/fisiologia , Fatores de Virulência/metabolismo , Acetamidas/química , Acetamidas/metabolismo , Acetamidas/farmacologia , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Sítios de Ligação , Domínio Catalítico , Modelos Animais de Doenças , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/genética , Microscopia Eletrônica de Transmissão , Simulação de Acoplamento Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Relação Estrutura-Atividade , Compostos de Sulfidrila/química , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/genéticaRESUMO
Onchocerciasis is an infection caused by the filarial worm Onchocerca volvulus, which can eventually result in blindness. The lack of an effective macrofilaricide and the possible development of ivermectin-resistant strains of O. volvulus necessitate the need for alternative treatment strategies. We have shown that targeting the L3-stage-specific chitinase OvCHT1 impairs the shedding of the filarial cuticle. In our continued efforts to discover OvCHT1 inhibitors, we identified the ß-carboline alkaloid scaffolding as a chitinase inhibitor that is capable of penetrating the worm cuticle. Herein, we disclose the rich polypharmacology of the ß-carboline class of compounds as an approach to abrogate the molting of the parasite and thus the initiation of infection in the human host.
RESUMO
The anthelmintic closantel has shown promise in abrogating the L3 molting of Onchocerca volvulus, the causative agent of the infectious disease onchocerciasis. In our search for alternative scaffolds, we utilized a fragment replacement/modification approach to generate novel chemotypes with improved chitinase inhibitory properties. Further evaluation of the compounds unveiled the potential of urea-tropolones as potent inhibitors of O. volvulus L3 molting.
RESUMO
The L3-stage-specific chitinase OvCHT1 has been implicated in the development of Onchocerca volvulus, the causative agent of onchocerciasis. Closantel, a known anthelmintic drug, was previously discovered as a potent and specific OvCHT1 inhibitor. As closantel is also a known protonophore, we performed a simple scaffold modulation to map out the structural features that are relevant for its individual or dual biochemical roles. Furthermore, we present that either OvCHT1 inhibition or protonophoric activity was capable of affecting O. volvulus L3 molting and that the presence of both activities in a single molecule yielded more potent inhibition of the nematode's developmental process.
Assuntos
Antinematódeos/uso terapêutico , Quitinases/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico , Ionóforos/uso terapêutico , Onchocerca volvulus/crescimento & desenvolvimento , Animais , Caenorhabditis elegans/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Muda/efeitos dos fármacos , Onchocerca volvulus/efeitos dos fármacos , Oncocercose/tratamento farmacológico , Salicilanilidas/química , Salicilanilidas/uso terapêutico , Relação Estrutura-Atividade , Desacopladores/uso terapêuticoRESUMO
A complication of diabetes is the inability of wounds to heal in diabetic patients. Diabetic wounds are refractory to healing due to the involvement of activated matrix metalloproteinases (MMPs), which remodel the tissue resulting in apoptosis. There are no readily available methods that identify active unregulated MMPs. With the use of a novel inhibitor-tethered resin that binds exclusively to the active forms of MMPs, coupled with proteomics, we quantified MMP-8 and MMP-9 in a mouse model of diabetic wounds. Topical treatment with a selective MMP-9 inhibitor led to acceleration of wound healing, re-epithelialization, and significantly attenuated apoptosis. In contrast, selective pharmacological inhibition of MMP-8 delayed wound healing, decreased re-epithelialization, and exhibited high apoptosis. The MMP-9 activity makes the wounds refractory to healing, whereas that of MMP-8 is beneficial. The treatment of diabetic wounds with a selective MMP-9 inhibitor holds great promise in providing heretofore-unavailable opportunities for intervention of this disease.
Assuntos
Complicações do Diabetes/tratamento farmacológico , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/uso terapêutico , Cicatrização/efeitos dos fármacos , Animais , Complicações do Diabetes/enzimologia , Complicações do Diabetes/patologia , Metaloproteinase 8 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Camundongos , Modelos MolecularesRESUMO
Traumatic brain injury (TBI) is a leading cause of death and long-term disability. Following the initial insult, severe TBI progresses to a secondary injury phase associated with biochemical and cellular changes. The secondary injury is thought to be responsible for the development of many of the neurological deficits observed after TBI and also provides a window of opportunity for therapeutic intervention. Matrix metalloproteinase-9 (MMP-9 or gelatinase B) expression is elevated in neurological diseases and its activation is an important factor in detrimental outcomes including excitotoxicity, mitochondrial dysfunction and apoptosis, and increases in inflammatory responses and astrogliosis. In this study, we used an experimental mouse model of TBI to examine the role of MMP-9 and the therapeutic potential of SB-3CT, a mechanism-based gelatinase selective inhibitor, in ameliorating the secondary injury. We observed that activation of MMP-9 occurred within one day following TBI, and remained elevated for 7 days after the initial insult. SB-3CT effectively attenuated MMP-9 activity, reduced brain lesion volumes and prevented neuronal loss and dendritic degeneration. Pharmacokinetic studies revealed that SB-3CT and its active metabolite, p-OH SB-3CT, were rapidly absorbed and distributed to the brain. Moreover, SB-3CT treatment mitigated microglial activation and astrogliosis after TBI. Importantly, SB-3CT treatment improved long-term neurobehavioral outcomes, including sensorimotor function, and hippocampus-associated spatial learning and memory. These results demonstrate that MMP-9 is a key target for therapy to attenuate secondary injury cascades and that this class of mechanism-based gelatinase inhibitor-with such desirable pharmacokinetic properties-holds considerable promise as a potential pharmacological treatment of TBI.
Assuntos
Lesões Encefálicas/patologia , Ativação Enzimática/efeitos dos fármacos , Compostos Heterocíclicos com 1 Anel/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Sulfonas/farmacologia , Análise de Variância , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Lesões Encefálicas/metabolismo , Fluorescência , Compostos Heterocíclicos com 1 Anel/farmacocinética , Técnicas Histológicas , Imuno-Histoquímica , Aprendizagem em Labirinto , Camundongos , Sulfonas/farmacocinéticaRESUMO
Brain metastasis occurs in 20-40% of cancer patients. Treatment is mostly palliative, and the inability of most drugs to penetrate the brain presents one of the greatest challenges in the development of therapeutics for brain metastasis. Matrix metalloproteinase-2 (MMP-2) plays important roles in invasion and vascularization of the central nervous system and represents a potential target for treatment of brain metastasis. Carbonate, O-phenyl carbamate, urea, and N-phenyl carbamate derivatives of SB-3CT, a selective and potent gelatinase inhibitor, were synthesized and evaluated. The O-phenyl carbamate and urea variants were selective and potent inhibitors of MMP-2. Carbamate 5b was metabolized to the potent gelatinase inhibitor 2, which was present at therapeutic concentrations in the brain. In contrast, phenyl urea 6b crossed the blood-brain barrier, however, higher doses would result in therapeutic brain concentrations. Carbamate 5b and urea 6b show potential for intervention of MMP-2-dependent diseases such as brain metastasis.
Assuntos
Barreira Hematoencefálica/metabolismo , Carbamatos/síntese química , Metaloproteinase 2 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/síntese química , Compostos de Fenilureia/síntese química , Sulfetos/síntese química , Animais , Área Sob a Curva , Carbamatos/química , Carbamatos/farmacocinética , Compostos Heterocíclicos com 1 Anel/síntese química , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/farmacocinética , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/farmacocinética , Camundongos , Modelos Químicos , Estrutura Molecular , Fenol/química , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacocinética , Relação Estrutura-Atividade , Sulfetos/química , Sulfetos/farmacocinéticaRESUMO
MMP-9 plays a detrimental role in the pathology of several neurological diseases and, thus, represents an important target for intervention. The water-soluble prodrug ND-478 is hydrolyzed to the active MMP-9 inhibitor ND-322, which in turn is N-acetylated to the even more potent metabolite ND-364. We used a sensitive bioanalytical method based on ultraperformance liquid chromatography with multiple-reaction monitoring detection to measure levels of ND-478, ND-322, and ND-364 in plasma and brain after administration of ND-478 and the metabolites. ND-478 did not cross the blood-brain barrier, as was expected; however the active metabolites ND-322 and ND-364 distributed to the brain. The active compound after administration of either ND-478 or ND-322 is likely ND-364. ND-322 is N-acetylated in both brain and liver, but it is so metabolized preferentially in liver. Since N-acetyltransferases involved in the metabolism of ND-322 to ND-364 are polymorphic, direct administration of the N-acetylated ND-364 would achieve the requisite therapeutic levels in the brain.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Química Encefálica , Metaloproteinase 9 da Matriz/farmacocinética , Inibidores de Metaloproteinases de Matriz/farmacocinética , Animais , Arginina/análogos & derivados , Arginina/análise , Arginina/farmacocinética , Cromatografia Líquida , Feminino , Espectrometria de Massas , Metaloproteinase 9 da Matriz/administração & dosagem , Metaloproteinase 9 da Matriz/análise , Inibidores de Metaloproteinases de Matriz/administração & dosagem , Inibidores de Metaloproteinases de Matriz/análise , Camundongos , Camundongos Endogâmicos C57BL , Sulfonas/análise , Sulfonas/farmacocinética , Distribuição TecidualRESUMO
SB-3CT, a potent and selective inhibitor of matrix metalloproteinase-2 and -9, has shown efficacy in several animal models of neurological diseases. One of the greatest challenges in the development of therapeutics for neurological diseases is the inability of drugs to cross the blood-brain barrier. A sensitive bioanalytical method based on ultraperformance liquid chromatography with multiple-reaction monitoring detection was developed to measure levels of SB-3CT, its active metabolite, the α-methyl analogue, and its p-hydroxy metabolite in plasma and brain. The compounds are rapidly absorbed and are readily distributed to the brain. The pharmacokinetic properties of these gelatinase inhibitors and the efficacy shown by SB-3CT in animal models of stroke, subarachnoid hemorrhage, and spinal cord injury indicate that this class of compounds holds considerable promise in the treatment of diseases of the central nervous system.
Assuntos
Barreira Hematoencefálica/metabolismo , Compostos Heterocíclicos com 1 Anel/metabolismo , Inibidores de Metaloproteinases de Matriz/metabolismo , Fármacos Neuroprotetores/metabolismo , Sulfonas/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Feminino , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/farmacologia , Masculino , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Sulfonas/química , Sulfonas/farmacologiaRESUMO
SB-3CT (1), a selective and potent thiirane-based gelatinase inhibitor, is effective in animal models of cancer metastasis and stroke; however, it is limited by poor aqueous solubility and extensive metabolism. We addressed these issues by blocking the primary site of metabolism and capitalizing on a prodrug strategy to achieve >5000-fold increased solubility. The amide prodrugs were quantitatively hydrolyzed in human blood to a potent gelatinase inhibitor, ND-322 (3). The arginyl amide prodrug (ND-478, 5d) was metabolically stable in mouse, rat, and human liver microsomes. Both 5d and 3 were nonmutagenic in the Ames II mutagenicity assay. The prodrug 5d showed moderate clearance of 0.0582 L/min/kg, remained mostly in the extracellular fluid compartment (Vd = 0.0978 L/kg), and had a terminal half-life of >4 h. The prodrug 5d had superior pharmacokinetic properties than those of 3, making the thiirane class of selective gelatinase inhibitors suitable for intravenous administration in the treatment of acute gelatinase-dependent diseases.
Assuntos
Arginina/análogos & derivados , Gelatinases/antagonistas & inibidores , Compostos Heterocíclicos com 1 Anel/síntese química , Pró-Fármacos/síntese química , Sulfonas/síntese química , Amidas/síntese química , Amidas/farmacocinética , Amidas/farmacologia , Animais , Arginina/síntese química , Arginina/farmacocinética , Arginina/farmacologia , Compostos Heterocíclicos com 1 Anel/farmacocinética , Compostos Heterocíclicos com 1 Anel/farmacologia , Humanos , Hidrólise , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Testes de Mutagenicidade , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Ratos , Solubilidade , Relação Estrutura-Atividade , Sulfonas/farmacocinética , Sulfonas/farmacologia , ÁguaRESUMO
Gelatinases (MMP-2 and MMP-9) have been implicated in a number of pathological conditions, including cancer and cardiovascular disease. Hence, small molecule inhibitors of these enzymes are highly sought for use as potential therapeutic agents. 2-(4-Phenoxyphenylsulfonylmethyl)thiirane (SB-3CT) has previously been demonstrated to be a potent and selective inhibitor of gelatinases, however, it is rapidly metabolized because of oxidation at the para position of the phenoxy ring and at the alpha-position to the sulfonyl group. alpha-Methyl variants of SB-3CT were conceived to improve metabolic stability and as mechanistic probes. We describe herein the synthesis and evaluation of these structural variants as potent inhibitors of gelatinases. Two (compounds 5b and 5d) among the four synthetic stereoisomers were found to exhibit slow-binding inhibition of gelatinases and MMP-14 (MT1-MMP), which is a hallmark of the mechanism of this class of inhibitors. The ability of these compounds to inhibit MMP-2, MMP-9, and MMP-14 could target cancer tissues more effectively. Metabolism of the newly synthesized inhibitors showed that both oxidation at the alpha-position to the sulfonyl group and oxidation at the para position of the terminal phenyl ring were prevented. Instead oxidation on the thiirane sulfur is the only biotransformation pathway observed for these gelatinase inhibitors.